This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Douglas, Ed J A Title: Characterisation of -lactam/Vaborbactam activity in Klebsiella pneumoniae General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Characterisation of β- lactam/Vaborbactam activity in Klebsiella pneumoniae Edward James Alexander Douglas Student number: 1405932 Cellular and Molecular Medicine MSc. (Res) A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Master of Science (Res) in the Faculty of Life Sciences, School of Cellular and Molecular Medicine Word count: 27425 ABSTRACT Carbapenem resistant Klebsiella pneumoniae is one of the most clinically relevant multidrug resistant pathogens, exacerbated by an ability to cause nosocomial infections. While treatment choices are limited, the introduction of two β-lactam/β-lactamase inhibitor combinations: meropenem/vaborbactam and ceftazidime/avibactam has revolutionised the management of cephalosporin and carbapenem resistant K. pneumoniae infections. With an increasing clinical dependence on these combination therapies, there is a need to study resistance mechanisms to allow prediction of what might be seen in the clinic and perhaps suggest ways to reverse resistance when it occurs. In particular, this project aims to better understand β-lactam/vaborbactam efficacy against laboratory grown bacteria and the mechanisms of resistance that emerge by generating mutants of K. pneumoniae clinical isolates with reduced susceptibility to β-lactams in combination with vaborbactam. We showed that vaborbactam is a potent inhibitor of class A and C β-lactamases in vivo but shows particular potency against bacteria carrying the class A, KPC carbapenemase, when used in combination with meropenem. Upon analysis of meropenem/vaborbactam resistant mutants, the outer membrane porin OmpK36 was frequently lost, as has been previously reported. We also identified a novel meropenem/vaborbactam resistance mechanism, which involves down-regulation of OmpK36 and the Maltoporin LamB2. An attempt was made to find a universal β-lactam/vaborbactam combination that would be clinically active against meropenem/vaborbactam resistant K. pneumoniae producing KPC, CTX-M or the OXA-48 like carbapenemase OXA-232. While imipenem/vaborbactam and ceftazidime/vaborbactam were active against meropenem/vaborbactam mutants of CTX-M and OXA-232 producers, an alternative combination could not be found that was active against a meropenem/vaborbactam resistant mutant derived from a KPC producer. Furthermore, ceftazidime/vaborbactam resistance was identified in CTX-M producers as being caused by over-production of the CTX-M enzyme. Imipenem/vaborbactam resistance was not identified in this background. Finally, demonstrated for the first time that combining avibactam/ceftazidime with meropenem/vaborbactam overcomes meropenem/vaborbactam resistance in all mutants generated. Therefore, we conclude that combining these β- lactam/β-lactamase inhibitor combinations in the same patient shows great potential in the treatment K. pneumoniae isolates resistant to one of these combinations ACKNOWLEDEGEMENTS I would firstly like to make a special thanks to my supervisor Dr Matthew Avison for giving me this amazing opportunity and welcoming me into your wonderful research team. You have provided me with invaluable experiences, guidance and advice throughout my degree. I am so grateful the amount of time you have been able to dedicate to, not only my research but also help in editing my thesis, you have been without a doubt the most supportive supervisor I could have hoped for. Additionally, the work environment and relationships forged in D60 is a testament to your supervision. Thank you for all the lab days out and Christmas lunches (and all the pints in the Colston arms). I will always try to remember to ‘Calm down big lad’. A big thank you also goes to my secondary supervisor Dr Jim Spencer, thank you for everything you have done for me, especially writing references for my PhD applications. To Dr Juan Carlos Jimenez Castellanos, Dr Jaqueline Findlay, Dr Yuiko Takebayashi, Dr Karina Calvopina Tapia, Dr Wan Nur Ismah Kamil, Dr Punyawee Dulyayangkul and John Shaw, you have all helped me so much, especially in showing me new laboratory techniques and sharing your expertise. You have been endlessly patient and have always shown me encouragement not only throughout my Master’s degree but also my Undergraduate project. All the cake from Catherine Tooke definitely helped as well. A big thank you to Dr Kate Heesom and the University of Bristol Proteomics Facility for the proteomic analysis. Proteomics has been a large part of my project, so without you this would not have been possible. To all members of the D60 lab, Dr Phil Hinchcliffe, Dr Charlotte Colenso, Victoria King, Eilis Braggington, Oliver Mounsey, Phoebe Cheung, Maryam Alzayn, Roberto Perez Chavarria, Emily Stevens, Emily Lythell, Alaa Alnahari, Zongfang Yang and Dina Atwiley thank you for all the support you have given me throughout my Undergraduate and Master’s degree. You have made the D60 working environment a thoroughly enjoyable experience. I will miss being part of this big family. To my mother Sue Douglas, my sisters Hannah Douglas and Gurpreet Douglas and my brothers William Douglas and Steven Langran, it’s been a hard couple of years for our family. But I could not have done this without your constant love and support. I love all of you so much. DEDICATION This Master’s thesis and the whole of my degree is dedicated to my father Colin Frederick Douglas (1957-2013) and my grandfather Frederick Douglas (1931-2018). Two great men loved and adored by everyone that knew them, taken far too soon. They will continue to be sorely missed by my grandmother Barbara, my mother Susan and my siblings Hannah and William. I hope I have made you both proud and that I continue to do so with the start of my PhD. I will continue to try and emulate and pass down your values and our proud family traditions. You have both taught me so much and been such amazing role models. I hope that I can become even a shadow of the men that you were. I miss you more every day. ‘Jamais arriere’ AUTHORS DECLARATION I declare that the work in this thesis was carried out in accordance with the requirements of the University’s Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in text, the work is the candidates own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. In addition, the e submission and hard copy are identical. SIGNED: EDWARD JAMES ALEXANDER DOUGLAS DATE 20/09/2018 CONTENTS PAGE Chapter 1: Introduction .................................................................................................................... 1 1.1. The History of Antibiotics ......................................................................................................... 2 1.2. Current Bacterial cell targets .................................................................................................. 3 1.3. Antibiotic Resistance ................................................................................................................ 4 1.4. β-lactams ................................................................................................................................... 7 1.4.1. Overview ............................................................................................................................. 7 1.4.2. β-lactam subgroups .......................................................................................................... 9 1.4.2.1. Penicillins .................................................................................................................... 9 1.4.2.2. Cephalosporins ........................................................................................................ 11 .................................................................................................................................................. 13 .................................................................................................................................................